Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract.Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut.  Streptococcus thermophilus is a lactic acid bacterium traditionally used by the food industry because of its ability to ferment and acidify milk. It is one of the two bacteria used in yogurt, defined as “the coagulated milk product obtained by lactic acid fermentation through the action of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and S. thermophilus from milk and milk products"  in the final product, with S. thermophilus being more abundant  and LMG18311  were used. Stock cultures of S. thermophilus LMD-9 and LMG18311 were prepared in reconstituted 10% (w/v) Nilac skim milk .The strains Lactococcus lactis bacterial strains were used in this study: L. lactis subsp. lactis TIL448 (NCDO2110), isolated from peas, and TIL1230, a derivative of TIL448 obtained after curing plasmids by acridine orange treatment, devoid of pili and mucus-binding protein (S. thermophilus) or 30°C (L. lactis) in M17-lactose [1% (w/v)] (S. thermophilus) or glucose [0.5% (w/v)] (L. lactis) medium (M17Glc). This preculture was then used to inoculate M17Glc at 37 or 30°C. Bacteria were harvested during the exponential growth phase [optical density at 600 nm (OD600) of approximately 3] by centrifugation  and the pellet was washed once with phosphate buffered saline (PBS).Two  protein . Bacterister_0152::kana strain (LMD-9Kana) was constructed as follows. The kanamycin (kana) cassette of the plasmid pKa  and A3 (5′GGACAGC CGTAAACTATC3′) primers for the upstream fragment, and A2 (5′AGGGGTCCCGAGCGCCTACGAGGAATTTGTATCGA T3GGGACAGAACATTGTACC3′) and A4 (5′CATCCATTAA GACGCCCC3′) primers for the downstream fragment. The 3′ end of the generated upstream fragment contained a sequence complementary to the 5′ end of the kana cassette, whereas the 5′ end of the generated downstream fragment contained a sequence complementary to the 3′ end of the cassette. This allowed joining of these three fragments after TaqPhusion PCR using primers A3 and A4. After purification with a QIAquick PCR purification kit, 500 ng of the resulting fragment was further used to transform competent natural LMD-9 cells as described by Kanaclones were selected on M17Lac plates with kanamycin (1 mg/ml) and then verified by two PCRs using oligonucleotides A1 × A3 and A2 × A4. Finally, sequencing of the flanking regions was performed to ensure that no unwanted mutations were introduced. The two strains, LMD-9Kana and native LMD-9, grew similarly in M17 lactose-rich  medium and milk (data not shown).LMD-9 smid pKa  was PCR S. thermophilus LMD-9 and LMG18311 to degrade mucin sugars was evaluated by following their growth in microtiter plates with Yeast Extract medium  containing 10 g/L of one of the carbon sources to be tested  at 42°C. The growth rates are expressed as μMaxmeans ± SD (n = 7).The ability of L-glutamine 200 mM, and 1% (v/v) penicillin-streptomycin mixture  . Cells were seeded at a concentration of 2.5 × 104 cells/cm2 in six-well tissue-culture plates . Two days before the experiments, antibiotics were no longer added to the cell culture medium. Experiments were carried out 20–22 days post seeding to ensure full differentiation of cells. Cells were maintained at 37°C in a humidified atmosphere with 10% CO2 and the culture medium was changed daily.The mucus-secreting cell line HT29-MTX was obtained from Thecla Lesuffleur . Cells were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) containing phenol red and 4.5 g/L glucose , supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum, 1% (v/v) S. thermophilus LMD-9 to the HT29-MTX cell line were performed according to the method described previously by 600nm = 0.05. After an adaptation phase, bacteria were washed, suspended in fresh culture medium with 2% FBS, and diluted to obtain a bacterial-cell concentration of 3–4 × 107CFU/mL. HT29-MTX cells were gently washed twice with PBS, pH 7.5 (Lonza) and 2 mL bacterial suspensions added to each well, resulting in a bacterial cell to epithelial cell ratio (MOI) of 10:1. The bacterial and epithelial cells were co-incubated for 2 h at 37°C in a humidified atmosphere with 10% CO2. After incubation, HT29-MTX cells were washed twice with PBS to remove unbound bacteria, scraped with 0.1% (v/v) Triton X-100 , filtered five times through a 21-gauge needle, and incubated for 30 min at room temperature. The number of viable bacterial cells was determined in the cell pellet  and bacterial suspension after incubation in empty wells (control input) by the plating method. Results are expressed as the percentage of adherent bacterial cells relative to the number of bacteria added (control input). At least three independent experiments were performed and at least three serial dilutions in duplicate were carried out for each well.Adhesion experiments of Cells were cultivated for 7 days and incubated with 0, 20, or 50 mM lactate  for 17 h. The pH was verified and adjusted to that of normal culture medium for culture medium containing lactate. Each condition was tested in triplicate and experiments were repeated three times. Cells were scraped off and immediately used for protein extraction.S. thermophilus LMD-9 and LMG18311 to PGM-coated polystyrene plates was tested using the technique described for the L. lactis IBB477 strain (600nm = 1) were incubated in coated plates for 3 h at room temperature, unbound bacteria washed away, and adherent bacteria stained with crystal violet before solubilization in acetic acid. Adhesion is expressed as the optical density (OD583nm) of stained cells. Bacterial adhesion was determined in three independent experiments and results are presented as the means ± SD. The average value of at least six measurements was calculated after rejecting extreme results.A solution of type III mucin from porcine stomach (PGM)  [10 mg/mL] was dissolved in phosphate-buffered saline (PBS) pH 7.5 and used to coat 96-well polystyrene microtiter plates (Thermo Fisher Scientific - Nunc A/S). Adhesion of 7 strain . TIL448 7 strain . BrieflyColorectal tissues from healthy individuals arised from patients with diverticulosis. The use of human tissues for this study was approved by the local hospital ethics committee and French Ministry of Higher Education and Research (DC-2008-242). All subjects gave written informed consent in accordance with the Declaration of Helsinki.S. thermophilus LMD-9 to human colonic mucin. TIL448 and TIL1230 were used as high-adhesive and low-adhesive controls, respectively  were stained with DAPI for 15 min at room temperature in the dark. Labeled bacteria were added to the membrane in blocking buffer. After incubation for 1 h at room temperature in the dark, the fluorescence of adherent bacteria was detected by a ChemiGenius 2 imaging system (Syngene).The procedure was adapted from that of ectively . In brieAll procedures for gnotobiotic rats were carried out according to European guidelines for the care and use of laboratory animals with permission from the French Veterinary Services dedicated to M. T (78-123) and as previously described by All mice experiments were approved by the local Animal Ethics Review Committee of the Faculty/Paris 7 University with permission dedicated to the unit of EOD [EU0543 - Fac Méd X. Bichat – Animal. Centr. - APAFiS - Autor. APAFiS #6424].8 CFU/mL S. thermophilus LMD-9 . As a control, GF rats (n = 5) were also inoculated with 1 mL sterile Nilac milk (without bacteria). All gnotobiotic rats for which the drinking water was supplemented with lactose (4.5%wt/vol) are designated as +lac . All rats were euthanized 30 days after gavage to recover biological samples.Ino-LMD9: All mono-colonized rats were previously described by Kana mice: Male C57BL/6J mice were obtained from Charles River Laboratories. After receipt, mice were acclimated for 1 week and then randomized into control (n = 5) and treated (n = 5) groups. Mice were housed in a conventional animal facility under a 12:12-h light/dark cycle. Food and tap water were provided ad libitum. Five individuals per cage were housed in the same room. At the age of 4 weeks, mice were inoculated by oral gavage (108 CFU/mL in 150 μL/mouse), five times a week (from Monday to Friday), at 10 a.m. for 3 weeks, either with S. thermophilus LMD9Kana  or Nilac skim milk  (n = 5). Stool samples were collected two times a week at 4 p.m. All mice were euthanized at the age of 7 weeks by cervical dislocation.Ino-LMD9® Plus slides . Paraffin-embedded sections were deparaffinized and stained with Hematoxylin and Alcian blue (AB) solution pH 2.5 to count the number of total and goblet cells per crypt, respectively. MUC2 immunostaining was performed for rats using the EnVision + System-HRP  and anti-MUC2 antibody , as previously described , dehydrated, and embedded in paraffin, according to standard histological protocols. Four-micrometer thick sections of rat distal colon  were mounted on SuperFrostescribed . Only U-Colonic epithelial cells were isolated from the whole colon of rats and mice according to the method of ® kit  and an Agilent 2100 bioanalyzer at the ICE platform . All RNA had a RIN between 8 and 10, indicating that the RNA of all samples was of high quality. Reverse transcription was performed with 7 μg RNA of each sample using the High-Capacity cDNA Archive Kit  according to the manufacturer’s instructions. No inhibition was detected for any sample using the TaqMan® Exogenous Internal Positive Control (Applied Biosystems). The cDNA products were analyzed in triplicate by RT-qPCR with an ABI PRISM 7000 Sequence Detection System and 7000 system software, version 1.2.3 (Applied Biosystems). Muc2 mRNA was analyzed using TaqMan Gene Expression Assays  was used as a reference. Results obtained were normalized to the value for 18S rRNA and compared with the mean target gene expression in GF rats as the calibrator sample. The following formula was used: fold change = 2−ΔΔCt, where ΔΔCt threshold cycle (Ct) equals (target Ct – reference Ct) of the sample minus (target Ct – reference Ct) of the calibrator.Total RNA was extracted from colonic epithelial cells isolated from rat tissues by the guanidinium thiocyanate method . RNA conAcetate, propionate, butyrate, valerate, isobutyrate, and isovalerate concentrations were determined in cecum contents of mice after water extraction of acidified samples by gas liquid chromatography , as previously described . Lactate+. The relative percent of each oligosaccharide was calculated based on the integration of peaks of MS spectra.Distal colonic mucosa was scraped and the mucins solubilized and purified by isopycnic density-gradient centrifugation  . The mucin vitro binding experiments, data are reported as the means ± standard errors of the means (SEM) and one-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison test, was performed to compare data groups. Asterisks indicate values significantly different from the high-adhesive L. lactis strain or from low-adhesive L. lactis stain (∗∗∗p < 0.001 and ∗∗p < 0.01). For animal experiments, data are reported as the means ± standard errors of the means (SEM) and t-test or ANOVA, followed by Tukey’s Student range test, were performed to compare the data between different batches of animals (∗p < 0.05).Statistical analysis was performed using GraphPad Prism software . For S. thermophilus to bind mucus and mucins. We used two types of purified mucins: commercial pig gastric mucin (PGM) and colonic mucin isolated from human biopsies. The LMD-9 strain was co-incubated with PGM coated on polystyrene microplates  or human mucin vs. PGM spotted on nitrocellulose membranes . The TIL448 L. lactis strain, known to be highly adhesive . The adhesive profile of L. lactis TIL448 was also high for human samples, whereas that of S. thermophilus LMD-9 was low, identical to that of TIL1230 . We also used intestinal HT29-MTX cells to mimic the continuous and dynamic production of mucus . Binding of the S. thermophilus LMD-9 strain to HT29-MTX cells was also low. Thus, S. thermophilus LMD-9 displayed poor binding to mucus and mucins relative to that of the high-adhesive L. lactis TIL448 strain, regardless of the model tested . The low ability of S. thermophilus to bind mucin was confirmed with the other strain tested, LMG18311 (data not shown). We then evaluated the ability of S. thermophilus to use the sugars forming the mucin glycans as their sole carbon source. The growth of S. thermophilus was characterized in microplates with culture medium supplemented with galactose, mannose, fucose, N-acetyl glucosamine, N-acetyl galactosamine, or lactose, which is the preferred substrate of S. thermophilus. The maximal growth rate (μmax) of LMD-9 was 0.27 ± 0.08 h−1 in the presence of lactose, whereas this strain did not grow in any of the sugars forming mucins as their sole carbon source. We obtained similar results with the LMG18311 strain, with a μmax reaching 0.30 ± 0.05 h−1 in the presence of lactose and no growth in the presence of the mucin sugars. Overall, these data show that two strains of S. thermophilus (LMD-9 and LMG18311) adhere poorly to mucus and mucins in vitro, and are unable to grow in vitro with mucin sugars as the sole carbon source.We established various models to assess the ability of adhesive , was useS. thermophilus to modulate the mucus pathway of intestinal epithelial cells by studying mono-colonized adult rats, harboring LMD-9 as the sole bacterium in their digestive tract. Ino-LMD9 rats, which we previously described . When lactose (4.5% w/v) was added in drinking water . The presence of S. thermophilus in the digestive tract induced the amount of KLF4 protein in epithelial cells of the colon in two groups without and with lactose . The level of induction is 1.9 (±0.6) in Ino-LMD9 and 2.1(±0.46) in Ino-LMD9+lac in comparison with their respective germ free counterparts (p < 0.05) and the two groups of mono-associated rats (with and without lactose) displayed similar amount of KLF4 protein. There was also a comparable induction of KLF4 in mono-colonized rats carrying the LMG18311 strain . Goblet cells stained by Alcian Blue were more abundant in Ino-LMD9+lac than with GF+lac rats , whereas the number of total cells per crypt was the same. The production of Muc2 protein, the main mucin secreted in the colon, seems to be higher in the Ino-LMD9+lac rats than in GF , but the mRNA level and the number of Muc2 positive cells were unchanged after colonization of the colon by S. thermophilus LMD9+lac . Overall, these data show that two strains of S. thermophilus, LMD9 and LMG18311, induced KLF4 protein in mono-colonized rats in the presence or not of a continuous lactose supply, concomitantly with the accumulation of high amount of lactate (from 13 to 50 mM) in their digestive tract. We then evaluated the effect of lactate (20 and 50 mM) on KLF4 protein production by the mucus-producing HT29-MTX cell line . Lactate significantly increased the amount of KLF4 and the level of induction was equivalent regardless of the concentration tested (20 or 50 mM in vitro). Overall, S. thermophilus can induce KLF4 protein and the number of Alcian-Blue positive goblet cells when it is the only bacterium in the digestive tract of rats. Based on our observations in HT29-MTX cell line, we can suppose that lactate may be involved in the induction of KLF4.We tested the ability of escribed , were cong water , the levS. thermophilus in mice harboring a complex microbiota by constructing an LMD-9 mutant strain resistant to kanamycin (LMD-9Kana), enabling the evaluation of the colonization level of S. thermophilus in a complex bacterial community. The adult mice received either milk fermented with LMD-9Kana or only milk (Ctrl) five times per week for 3 weeks . We detected 8–9 103 CFU/g feces of LMD-9Kana , showing that live bacteria transited through the digestive tract, whereas no LMD-9Kana was present in the Ctrl group, as expected. We assessed the fermentative profile of the microbial community by measuring the levels of short chain fatty acids and lactate in the cecum . Mice receiving LMD-9Kana had significant levels of lactate (up to 0.5 mM) in the cecum and lower levels of propionate, isobutyrate, and isovalerate than the Ctrl group (p < 0.05). Thus, the transiting of S. thermophilus as sub-dominant bacteria, may orientate the activity of the resident and abundant microbiota. The number of goblet cells and KLF4 protein levels in the colon were not different in the mice that received LMD-9Kana than in Ctrl mice . Probiotic or commensal bacteria are known to modulate the composition and physical properties of mucus through changes in mucin O-glycans . Thus, transit of S. thermophilus as a sub-dominant bacterium through the microbiota resulted in modification of some bacterial metabolite levels, with lactate levels, no induction of the mucus pathway, and a moderate induction of sulfated mucin.The use of mono-colonized rodents allowed us to assess several mucus-related functions in an over-simplified digestive system. We next characterized the effect of -glycans . We thusS. thermophilus and mucus, using a battery of tests on in vitro and in vivo models. Two strains of S. thermophilus, LMD-9 and LMG18311, poorly adhered to mucus and mucins  and were unable to degrade various sugars used in mucin synthesis. S. thermophilus is thus a poor adhesive bacterium relative to other well-described mucus-adhesive lactic-acid bacteria, such as Lactobacillus reuteri and Lactobacillus plantarum , equivalent to the amount of S. thermophilus in ≈50 g of yogurt. In mice, we have administrated 5 times per week for 3 weeks 0.15 ml of pure S. thermophilus culture of 108 CFU/ml, equivalent to ≈1.5 g of yogurt per gavage.We have administrated S. thermophilus is the only bacterial species in the digestive tract . Mucus reinforcement is one of the mechanisms responsible for the beneficial effects of several lactic-acid bacteria. For example, Lactobacillus rhamnosus GG up-regulates mucin gene expression and mucus production in intestinal epithelial cell lines through two actors: p40-derived soluble protein of the bacteria and the EGF receptor of host cells  suggest that lactate produced by S. thermophilus in gnotobiotic model may sustain at least in part the stimulation of KLF4, involved in differentiation of secretive cells. This is consistent with the stimulation of cell-cycle arrest proteins and the anti-proliferative effect described for lactate in vivo and in vitro . Regular gavage of these mice with S. thermophilus LMD-9Kana did not alter the number of goblet cells nor KLF4 protein levels in the colon  or ileum (data not shown). The O-glycosylation pattern of mucins plays an important role in intestinal homeostasis  and lactate was detectable in the cecum at higher levels than in non-treated animals, although the difference was small. Under normal conditions, lactate does not accumulate in the intestinal lumen of adults, because other bacteria consume it .Supplementation of conventional mice with nsume it . Higher nsume it . In the r jejuni . A lactar jejuni  and atter jejuni . TherefoS. thermophilus adheres poorly to mucus and poorly degrades mucin sugars in vitro. We propose that lactate, produced in the gut by S. thermophilus may potentially induce pathways involved in mucus induction, as revealed using gnotobiotic rodents to study the novel functions of transient food-borne bacteria. This suggests that the consumption of fermented dairy products may reinforce mucosal integrity of the intestine. Although we did not observe mucus induction in a more sophisticated physiological context with a complex microbiota, we believe that this newly revealed S. thermophilus-driven function may be protective under certain conditions in which the mucus barrier is altered.Overall, the lactose-degrading probiotic MC-B, FR, EO-D, MT, and MM-B designed the project, obtained the financial support, and supervised the experiments. NF, LW, JR-B, BR-D, M-PD, M-LN, VL, VR, CC, M-LD-M, RL, and CR-M produced and/or analyzed the results. MT and MM-B wrote the article. All the authors have read and approved the last version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using voxel-based morphometry (VBM), we additionally determined the structural correlates of reward-related motor performance (RMP) and PL. Monetary reward had a beneficial effect on PL in the older subjects only. In contrast, social reward significantly enhanced PL in the older and consolidation in the young participants. VBM analyses revealed that motor performance related to monetary reward was associated with larger grey matter volume (GMV) of the left striatum in the young, and motor performance related to social reward with larger GMV of the medial orbitofrontal cortex in the older group. The differential effects of social reward in young (improved consolidation) and both social and monetary rewards in older (enhanced PL) healthy subjects point to the potential of rewards for interventions targeting aging-associated motor decline or stroke-induced motor deficits.In young ( The preservation of acquired as well as the acquisition of new motor skills are essential across the entire lifespan. In particular, when aging-associated impairments of motor function occur or when aging-associated diseases, e.g., a stroke, impact upon the motor system, training of previously learnt motor skills or the acquisition of new motor skills become necessary. Given the sociodemographic changes and the resulting increasing incidence of stroke , a deepeOne way to enhance procedural learning and consolidation, the process leading to the retention of the learnt motor knowledge , is the Unfortunately, most studies investigated young participants only. A recent study has shown that the application of monetary reward can support procedural learning and retention in patients with stroke . ConcernTaken together, these previous findings suggest age-dependent effects of social and monetary reward on procedural learning and consolidation. Accordingly, we compared these two kinds of rewards in younger and older subjects. Putative differential, age-related effects of social and monetary rewards on procedural learning and consolidation might eventually be used to counteract aging- or disease-associated impairments of motor function.To investigate the specific effects of reward on procedural learning and consolidation in aging, we used a modified version of the serial reaction time (SRT) task . AccordiAnatomical studies suggest an association between regional brain volumes and the performance in motor tasks  as well n = 36, 18 men; age (mean ± SD): 24.8 ± 4.5 years, range: 18–35 years) and one with older participants (n = 36). Exclusion criteria were any history of a neurological or psychiatric disorder or the use of psychopharmacologically active medication. In the group of older subjects, one subject had to be excluded from further analysis due to technical issues, another one due to an error rate >3 standard deviations from the mean of the older group. Thus, data from 34 older subjects entered the subsequent analyses . Structural MRI scans were acquired for a subset of 28 young and 32 older participants. Using Oldfield’s laterality quotient  and all (young and older) participants filled out the Beck Depression Inventory (BDI) to rule out clinically relevant symptoms of depression as well as the German version of the Barratt Impulsiveness Scale (BIS-11) . Each paAll subjects gave written informed consent before their participation. The ethics committee of the Faculty of Medicine, University of Cologne, had approved the study.® . Subjects were instructed to press the spatially congruent button on a custom-made three-button-keyboard as fast as possible. When the response in the trial was correct and the reaction time (RT) was lower than the individual median RT , a feedback was shown for 250 ms followed by an inter-stimulus interval of 250 ms. The median RT of the practice session was used to reduce the impact of potential outliers  on the criterion that triggered the application of reward/feedback in the experimental blocks.We used a modified version of the Serial Reaction Time (SRT) Task . SubjectWe used three types of feedback: a monetary reward, a social reward, and a neutral reward/feedback (for control). In the monetary reward condition, a 50 € banknote was displayed at the centre of the screen. Participants were informed that every time they saw the 50 € note, their pay would increase by 0.05 €. In the social reward condition, a photo of one investigator  giving positive feedback was shown . The useStimuli were presented either in a repetitive sequential manner (S) or a pseudo-random (R) succession, but subjects were not informed about the presence of a sequential pattern. Three different 9-element sequences were used; one for each reward/feedback condition . Each reward/feedback condition consisted of seven blocks: R1-S2-S3-S4-s5-r6-s7. During the first four blocks (R1–S4) reward/feedback was presented to promote reward-based procedural learning. The last three blocks  were used to assess the sequence-specific learning effect. To avoid a confound of reward/feedback on the retrieval of the learnt sequences, we chose not to apply reward/feedback during these three blocks. We added block s7 to rule out that fatigue effects provoked a potential increase in RT between the blocks used for calculation of the sequence-specific learning effect . Within the five sequence blocks , each 9-element sequence was repeated five times per block, which resulted in twenty-five repetitions of each sequence in total. Breaks separated the blocks. The subjects could freely choose the length of the break by pressing the enter key to continue with the next block. The order of reward/feedback conditions was counterbalanced across subjects. This means that the three reward/feedback conditions were performed in separate sessions, each consisting of seven blocks . Every possible combination of reward/feedback conditions  and sequences  was equally distributed across subjects. Each given combination was performed by six subjects, except the combinations where participants had to be excluded as mentioned above.On day 2, subjects performed a shortened version of the same task  and without any feedback, using the same three sequences as on day 1  in the same order as on day 1 to investigate consolidation. In contrast to day 1 where multiple “sequence blocks” were repeated, we chose this alternating structure of blocks to avoid further sequence-specific learning on day 2 but rather concentrated on assessing consolidation. We used multiple “random blocks” to rule out RT effects caused by fatigue or general practice.We used three different 9-element sequences: 2-1-3-3-2-2-3-1-1, 3-2-3-3-1-2-2-1-1, and 3-3-1-3-2-2-1-1-2 . All sequences – including the ones used in the r/R blocks – were constructed according to the following rules as applied in previous studies  to avoid(1)No more than two repeats of one stimulus location .(2)No more than two unidirectional stimulus locations in a row .(3)No repeated doublets .(4)No three repetitions in a row .(5)Each stimulus location had to appear three times in every sequence.(6)Each stimulus location occurred three times, each time followed by a different stimulus location.In other words, the frequency of all possible stimulus locations and the transition probabilities between consecutive stimulus locations were equally distributed across sequences.After completion of the task, explicit motor sequence knowledge was assessed on day 2 using a standardised structured interview and a free recall test. First, we asked the participants whether they had recognised something about the experiment that we had not told them. Secondly, if the participants did not report any irregularities, we asked the participants whether they had recognised something about the succession of the stimuli. If participants stated that they had not recognised anything about the succession of the stimuli, we asked the participants whether they thought that the succession had been random or not. Participants who still described the succession as being random were told about the underlying structure of the experiment. All participants were then asked to reproduce at least one of the sequences used (by ticking off the positions of the stimuli on a sheet of paper) and to assign the associated reward condition. Participants were allowed to reproduce as many of the three sequences as they wished. We operationalized explicit motor sequence knowledge as the highest count of correct items in a row.® 21 (IBM) and RStudio 0.98 were used to analyse the behavioural data. The first trial of each block was rejected. Furthermore, error trials and the respective following trial were removed, and the median RT per block was calculated for every subject. Again, the median RT per block was used to reduce the impact of potential outliers . These were then used to calculate the mean RT across subjects for every block. Similar to previous studies investigating procedural learning  suggesting that RMP represents not a mere improvement in task performance or motor sequence knowledge. As there is evidence for an association between reward sensitivity and impulsiveness in an event-related potentials (ERP) study .We operationalized motor performance related to monetary and social rewards as the percentage of rewarded trials of each participant in the monetary and the social reward conditions, respectively. A correlation analysis between reward-related motor performance (RMP) and general as well as sequence-specific learning for both groups revealed no significant correlations  study , we furtMagnetic resonance imaging was conducted on a 3.0 T Trio scanner  using a 12-channel Siemens head coil. The structural T1-weighted magnetisation prepared gradient-echo images were acquired using the following parameters: repetition time (TR) 2250 ms; echo time (TE) 3.03 ms; field of view (FOV) 256 mm; 176 sagittal slices of 1 mm thickness; flip angle = 9°, voxel size 1 × 1 × 1 mm. We screened MR images visually for artefacts and to exclude pathology.1. Subsequently, the VBM8 toolbox 2 was employed using the default parameters. Images were first reoriented and aligned to the anterior commissure, followed by segmentation into grey matter (GM), white matter (WM), and cerebrospinal fluid (CSF). The resulting images were normalised to the stereotactic space of the Montreal Neurological Institute (MNI) using the iterative high-dimensional “Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra” (DARTEL) normalisation procedure. As spatial normalisation expands and contracts some brain regions, modulation was performed subsequently. This involves scaling by the amount of concentration so that the total amount of GM in the modulated GM remains the same as it was in the original images. Checking sample homogeneity resulted in the exclusion of one older participant. Modulated GM segments of 30 older and 28 younger participants were then smoothed using a Gaussian kernel with a value of 10-mm full-width as recommended by the VBM8-Toolbox Manual.Data were processed and analysed using the Statistical Parametric Mapping software t-test with the smoothed GM images to investigate the association of GMV with the participants’ behavioural measures.Voxel-based morphometry (VBM) was used to examine the neural correlate of RMP and the susceptibility to a given type of feedback during procedural learning at different ages. We, therefore, assessed the relation of these parameters with GMV for both age groups separately. The behavioural data of young and older participants were split into terciles depending on the percentage of rewarded trials (RMP) or the SSL effect  the participants achieved in each reward condition on day 1. Separately for young and older participants, we then compared the GMV of participants in the first tercile  with the GMV of the respective participants in the third tercile . The same was also done for the SSL effects on day 1. Using SPM8, we conducted a two-sample x, y, z = −6, 36, −15) was defined according to functional imaging data by Lin and colleagues  were defined according to a stereotactic investigation .Based on previous studies , we perflleagues . The ROItigation . The WFUtigation , respecttigation . The thrt(66) = 6.373; p < 0.001]. Neither group showed clinically relevant symptoms of depression in the BDI (cut-off > 9) . Moreover, the groups did not differ concerning impulsiveness, according to the Barratt Impulsiveness Scale (t(64) = 0.274; p = 0.785). All older subjects achieved unremarkable scores in the dementia screening test MMSE.The two groups showed a significantly different performance in the Corsi block-tapping test [young subjects: 6.42 ± 1.08 (mean ± SD); older subjects: 4.97 ± 0.74; All descriptive statistics are provided as mean ± standard error of the mean (SEM) unless stated otherwise.t(68) = 1.345; p = 0.183; day 2: t(68) = 1.286; p = 0.203). Moreover, the mean error rates across conditions were <1% for both groups . Due to this fact, we decided to restrict further analyses to reaction time (RT) data.On both days, there was no significant difference in error rates between groups (day 1: t(68) = 7.670; p < 0.001).As expected, young participants had significantly faster mean RT  = 2.167; p < 0.05) and SSL  = 4.443; p < 0.001) effect. On day 2, the significant SSL effect had been retained in the young group  = 3.814; p < 0.01). The same applied to the group of older participants  = 2.815; p < 0.01; SSL on day 1: 15.7 ± 5.3 ms; t(33) = 2.990; p < 0.01; SSL on day 2: 10.3 ± 3.5 ms; t(33) = 2.968; p < 0.01).On day 1, irrespective of reward type, younger participants showed a significant GL  = 1.449; p = 0.242; older: F = 1.339; p = 0.269).Concerning GL on day 1, neither group showed a differential effect of reward  = 2.284; p = 0.109) was detected for the young group  = 3.409; p < 0.05). Post hoc tests revealed a significantly higher SSL effect for social compared to neutral reward (t(33) = 2.554; p < 0.05, one-sided, Bonferroni-corrected) and for monetary vs. neutral reward (t(33) = 2.242; p < 0.05, one-sided, Bonferroni-corrected). SSL effects for monetary and social rewards on day 1 did not differ significantly in the older group (t(33) = 0.247, p = 0.8; For SSL on day 1, no significant differential influence of reward type  = 0.975, p = 0.380) or a significant main effect of order  = 0.105, p = 0.900), nor for sequence-specific learning  = 1.333, p = 0.267; main effect of order: F = 0.170, p = 0.844).We performed repeated-measures ANOVAs for general learning and sequence-specific learning on day 1 with the within-subject factor order of sequences/rewards and the between-subject factor group to assess potential order effects on the efficiency of the reward conditions. Neither for general learning there was a significant interaction  = 14.703; p < 0.001) and group  = 15.803; p < 0.001). The latter resulted from a significantly higher proportion of rewarded trials in young compared to older participants. A comparison of the three reward types using post hoc t-tests revealed that the young group showed the highest proportion of rewarded trials in the monetary reward condition  = 5.760; p < 0.001, Bonferroni-corrected; vs. social reward: 76.4 ± 2.3%; t(35) = 2.495; n. s.), followed by the social reward condition  = 2.625; p < 0.05, Bonferroni-corrected). The older group presented the same tendency, however, only the proportion of rewarded trials in the monetary reward condition was significantly higher than the one in the neutral feedback condition  = 2.575; p < 0.05, Bonferroni-corrected).To analyse the RMP of the participants, we calculated the rate of rewarded trials separately for the three reward conditions on day 1. Repeated-measures ANOVAs with the within-subject factor reward  and the between-subject factor group revealed significant main effects of reward  = 0.428; p = 0.654; F = 8.122; p = 0.001; post hoc t-tests revealed that consolidation of the motor sequence knowledge learnt under social reward was significantly higher than that associated with neutral feedback (t(35) = 3.651; p < 0.01) or monetary reward (t(35) = 2.879; p < 0.01). Consolidation of motor sequence knowledge learnt under monetary reward did not differ from consolidation associated with neutral feedback (t(35) = 1.105; p = 0.139, all Bonferroni-corrected).The effects of the different types of reward on consolidation, operationalized by SSL on day 2, did no longer reach significance in the older participants  = 4.717; p = 0.010). This interaction was driven by the fact that only in the young group, there was a specific enhancement of the SSL effect from day 1 to day 2 for the sequences learnt under social reward (t(35) = 3.270; p < 0.01, Bonferroni-corrected). The main effects of group  = 0.025; p = 0.874), day  = 0.625; p = 0.432) and reward  = 2.959; p = 0.55) as well as all two-way interactions were not significant (p > 0.05).To assess potential changes in SSL from day 1 to day 2 and the effects of age and reward thereon, we performed a mixed-design ANOVA with the between-subjects factor group (older vs. young) and the within-subjects factors day (day 1 vs. day 2) and reward . There was a significant three-way interaction day × reward × group  = 6.279; p < 0.01), as SSL for the first sequence learnt was significantly larger than SSL for the third sequence (one-sided post hoc t-test: t(33) = 2.914; p < 0.01, Bonferroni-corrected, see p = 0.182).To elucidate potential causes for the lack of differential effects of reward on consolidation in the older participants, we analysed SSL on day 2 with a different approach. To this end, we used the order of sequences on day 1 (irrespective of the type of reward associated with a given sequence) as the independent variable. Especially in the older group, consolidation critically depended on the order of sequences on day 1 (t(66) = 0.100; p = 0.921), with a mean of 4.5 elements in each group . To assess a potential association between explicit motor sequence knowledge and SSL (on day 1), we performed a correlation analysis between the individual maximum number of correctly reproduced elements of the sequence in a row and the individual SSL. Only for the SSL in the neutral feedback condition, there was a significant, but negative correlation in the older group  indicating that the SSL of older subjects in the neutral feedback condition was lower when they achieved more explicit motor sequence knowledge. All other associations were not significant. Despite the notion that explicit motor sequence knowledge is an essential factor for motor/procedural learning . In contrast, better motor performance related to social reward correlated with a larger GMV in the mOFC of older subjects  as well as Ncl. accumbens and amygdala bilaterally . Note that no reward/feedback was given during the retrieval session on day 2. These findings are consistent with data providing evidence for an enhancement of consolidation by social reward in healthy young subjects . FurtherStudies investigating the effects of aging on motor performance reported a specific deterioration of consolidation in older subjects . NeuroimVBM revealed that RMP was differentially associated with increased GMV in two main regions of the reward system: whereas motor performance related to monetary reward was associated with increased GMV in the striatum of the young, motor performance related to social reward correlated with increased GMV in the mOFC of older subjects. The lack of a significant correlation between RMP and SSL or GL measures indicates that this parameter does not merely reflect a general ability to improve performance. Thus, the current results add to previous evidence for an association between personality traits and brain structure . SpecifiThe activity in the retention interval, the length of the interval, and the daytime of testing represent critical factors for consolidation . In the In conclusion, our data revealed age-dependent differential effects of social reward on motor performance: whereas social (and monetary) reward especially improved motor sequence learning in older participants, the consolidation of motor sequence knowledge was improved by social reward in young subjects only. These differential effects of social reward (on consolidation) in young and of both social and monetary reward  in older subjects point to the potential benefit of rewards for interventions counteracting aging- or disease-related motor deficits, an issue which warrants further investigation.This study was carried out in accordance with the recommendations of the ethics committee of the Faculty of Medicine, University of Cologne, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the Faculty of Medicine, University of Cologne.CD and LM conceived and designed the study, acquired the data, analysed the data, and drafted the manuscript. AD analysed the data and critically revised the manuscript. JS-B acquired the data. PW and GF conceived and designed the study, analysed the data, and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eggshell membranes, the thin film lining the insides of eggshells, are constituted mostly from protein . The digestibility and dietary net protein utility of ESM-P are not known. ESM-P functions as a barrier to prevent foreign matter from reaching the egg white and yolk, so it would be expected not to decompose easily by digestion when ingested. We therefore prepared a hydrolysate of the membrane (ESM-H). In this study, we assessed the digestibility and net protein utility of ESM-P and ESM-H in rats.The digestibility of ESM-P and ESM-H were 87.0% and 94.8%, respectively, significantly lower than that of casein (98.5%). The net protein utility values were 84.7% and 84.6%, respectively, significantly higher than that of casein (75.1%). Digestibility was significantly higher for ESM-H than for ESM-P, but there was no significant difference in net protein utility between ESM-P and ESM-H. These results demonstrated that more than 80% of ESM-P or ESM-H is absorbed and utilised in the bodies of rats. The eggshells and eggshell membranes of hens’ eggs are little used potential dietary resources. Eggshells are known to be a source of calcium, which can increase bone density . The eggCollagen is another protein reported to have a skin-moisturising effect and to ameliorate knee joint pain. Collagen is known not to be utilised in vivo independently because it contains little tryptophan . It has It has been reported that ESM-P include collagen type I, V, and X, and protein derived from egg white , 9. The The aim of this study was to assess the digestibility and net protein utility of ESM-P. Because ESM-P functions as barrier to prevent foreign matter from reaching the egg white and yolk, we thought it likely that the protein would not be easily decomposed by digestion when ingested. We therefore prepared a hydrolysate of eggshell membrane (ESM-H), using enzymatic hydrolysis to convert it to low molecular weight peptides, and assessed its digestibility and net protein utility in addition to those of ESM-P.Casein was purchased from Oriental Yeast Co., Ltd. (Tokyo). ESM-P and ESM-H were obtained from Kewpie Corporation (Tokyo). ESM-H was prepared by the alkaline protease digestion of ESM-P. After enzyme inactivation at 90 °C, the hydrolysate was spray-dried ready for testing . The amiEight-week-old male Sprague Dawley rats (245–275 g) were used (n = 6). The animals were kept in metabolic cages  at 23.1 °C, with 50% ± 2% humidity and a 12-h light (8:00–20:00)/dark cycle.2 gas.Test diets were prepared according to the American Institute of Nutrition (AIN)-76 formulation . They inThis experiment was conducted in accordance with the Guidelines for Animal Experiments, Law No. 105 and Notification No. 6, of the Government of Japan and the animal experiment rules of the Research and Development Headquarters. This experiment was approved by Ethics committee of Kewpie corporation R&D division (Reference and Permission No. 17-06). The experiment was performed from 5 to 22 December 2017.Faecal and urinary protein contents were calculated by measuring the nitrogen (N) content using the Dumas method  and multp value was less than 5%.Test results are expressed as mean ± SE. The statistical analyses used Tukey’s test and were performed using SPSS ver. 20 software . Differences were considered statistically significant when the Table Table Figure The results of this study demonstrated that more than 80% of ESM-P is absorbed and utilised in vivo. Follow-up test results showed that the digestibility and net protein utility of ESM-P were 88.8% and 82.4%, and those of ESM-H were 95.1% and 84.7%, respectively (unpublished data). These values were similar to the results of the present study, confirming its reproducibility.The digestibility and net protein utility values of ESM-P and ESM-H were lower than representative values for white proteins . This maAlthough total nitrogen excretion  of the casein group was higher than that of the ESM-P and ESM-H groups, the body weight of the casein group was higher than that of the ESM-P and ESM-H groups Table , Fig. 1.We believe that total nitrogen excretion and NPU affects the body’s protein content. Except for water content, animal bodies consist mainly of protein and fat. It has been reported that egg white protein increases the amount of body protein and decreases the amount of body fat compared with casein in rats . ESM incESM-P has been reported to have a skin-moisturising effect and to ameliorate knee joint pain , 6. SimiHyaluronic acid is a saccharide, but there are proteins that are known to exert similar effects. Collagen has been reported to have similar effects to those of ESM-P. However, collagen can hardly be absorbed independently because of its low content of tryptophan, an essential amino acid. Collagen contains abundant hydroxyproline , whereasIn the present study, urine volume was lower in the ESM-P group rats (26.3 ± 8.4 mL/5 days) compared with the volumes for those fed ESM-H and casein. Dietary intake and net protein utilisation were same in the ESM-P and ESM-H groups, but body weight was lower in the ESM-P group than in the ESM-H. Thus, the reason for the lower urine volume in the ESM-P group may have been lower body water content and water intake in this group compared with the ESM-H group. In a previous study, we found a similar urine volume in rats fed milk whey (27.3 ± 4.3 mL/5 days) . In thatThere are subjects in utilisation of ESM-P in foods, etc. expecting its functions for health. For use in foods, it may be necessary to digest eggshell membrane into low molecular weight molecules for efficient absorption, given its lower digestibility than casein , and to In this study, ESM-H showed higher digestibility than ESM-P, but their net protein utility was nearly the same. It is therefore likely that their net protein utility is influenced by their amino acid composition rather than their digestibility. The potential use of ESM-P and ESM-H in food and health products has been little studied. The results of this study should contribute to consideration of ESM-P and ESM-H as a new functional food ingredient.Clarification of this mechanism is likely to require an assessment of ESM-P biokinetics. It would be necessary first to assess the absorbability and receptor-binding capacity of ESM-P in the small intestine, for example, to establish its action in the digestive tract.


To estimate the prevalence of non-calcified coronary artery disease (CAD) in patients with suspected stable angina and a zero coronary artery calcification (CAC) score, and to assess the prognostic significance of a zero CAC in these symptomatic patients.In this prospective cohort study, consecutive patients with stable chest pain underwent CAC scoring ± CT coronary angiography (CTCA) as part of routine clinical care at a single tertiary centre over 7 years. Major adverse cardiac event (MACE) was defined as cardiac death, non-fatal myocardial infarction and/or non-elective revascularisation.A total of 915 of 1753 (52.2%) patients  had a zero CAC score. Of the 751 (82.1%) patients with a zero CAC in whom CTCA was performed, 674 (89.7%) had normal coronary arteries, 63 (8.4%) had non-calcified CAD with < 50% stenosis and 14 (1.9%) had ≥ 50% stenosis in at least one coronary artery. The negative predictive value of a zero CAC for excluding a ≥ 50% CTCA stenosis was 98.1%. Over a median follow-up period of 2.2 years (range 1.0–7.0 years), the absolute annualised rates of MACE were as follows: zero CAC 1.9 per 1000 person-years and non-zero CAC 7.4 per 1000 person-years . However, after adjusting for age, gender and cardiovascular risk factors using a multivariable Cox proportional hazards model, there was no statistically significant difference in the risk of MACE between the two patient cohorts (p = 0.19). After adjusting for age, gender and cardiovascular risk factors, the HR for all-cause mortality among the zero CAC cohort vers non-zero CAC was 2.1 (p = 0.27).A zero CAC score in patients undergoing CT scanning for suspected stable angina has a high negative predictive value for the exclusion of obstructive CAD and is associated with a good medium-term prognosis. Many existing studies have looked at the prognostic value of a zero calcium score in asymptomatic study populations which have different prior probabilities of risk of major adverse cardiac event compared with symptomatic patient populations.This study has the advantage of being a prospective study and looks at the medium-term prognosis of the symptomatic patient population with a zero calcium score.The discovery of underlying coronary artery disease in those with zero calcium scores highlights the advantages of using CT coronary angiography in all subjects rather than having calcium scoring as a gatekeeper, as was the case in the previous version of the National Institute for Health and Care Excellence CG95 guideline.Coronary artery calcification (CAC) is a well-established marker of future cardiovascular risk.2The absence of CAC (defined as a zero CAC score on CT) in asymptomatic individuals is associated with a very low incidence of cardiovascular events over a 15-year follow-up.6In November 2016, the UK’s National Institute for Health and Care Excellence’s (NICE) Clinical Guideline 95 for the evaluation of patients with stable chest pain was issued as an update of their own 2010 Guideline.Therefore, the objective of the study was to estimate the prevalence of non-calcified CAD in patients presenting with stable angina and a zero CAC score undergoing CT scanning in our centre. The secondary objectives were to compare incidence rates of major adverse cardiac events (MACEs), and all-cause mortality, in patients with a zero CAC versus a non-zero CAC score.In this prospective study, consecutive patients with a suspected new diagnosis of stable angina underwent CAC scoring ± CT coronary angiography (CTCA) as part of routine clinical care at Addenbrooke’s Hospital from November 2009 to October 2016. This was a service evaluation audit and it was approved by the hospital’s audit committee. Patients were excluded from the study if they were asymptomatic, had prior diagnosis of CAD, myocardial infarction, percutaneous coronary intervention or coronary artery bypass grafting surgery. Patients referred for a cardiac CT scan for non-coronary indications such as arrhythmias, aortic valve and pericardial evaluations were excluded.Clinical data were obtained from the hospital electronic patient record system. Hypertension was defined by a physician diagnosis of hypertension or the use of anti-hypertensive medications. Diabetes was defined by a physician diagnosis of diabetes or the use of anti-diabetic medications. Obesity was defined by a body mass index of 30 or greater. A positive smoking history was defined by the patient either being a current smoker (being an active smoker at the time of scan) or being an ex-smoker. A family history of premature CAD was defined as any first-degree relative with a history of relevant heart disease before age 60 or based on having a recorded family history of CAD in the patient record.Imaging was performed using a Siemens Flash 64×2-slice dual source CT scanner as per standard clinical protocols. CAC scoring was performed using the Agatston methodPatients were followed up prospectively using the hospital electronic patient record system.The endpoint, MACE, was defined as cardiac death, non-fatal myocardial infarction and/or non-elective revascularisation. Additionally, the incidence of all-cause mortality was evaluated. Revascularisation was defined as percutaneous coronary intervention or coronary artery bypass grafting surgery. A diagnosis of myocardial infarction was verified by a cardiologist.2, two-tailed test was used for comparison of categorical variables.Continuous variables are presented as mean±SD or median and IQR, and categorical variables as counts with proportions. Comparison of between-group continuous variables was performed using the unpaired t-test, whereas the Pearson χA survival analysis was conducted with individual subject time-to-MACE using the Kaplan-Meier method, with curves representing MACE-free survival according to zero CAC or non-zero CAC. Annualised rates of MACE and all-cause mortality were estimated by dividing the number of events by the number of person-years at risk.HR and 95% CIs were calculated with the Cox proportional hazards regression model using zero CAC as the reference group. Three models were constructed: Model 1: unadjusted, univariable analysis; Model 2: multivariable analysis adjusting for age and gender; Model 3: multivariable analysis adjusting for age, gender and cardiovascular risk factors .All statistical analyses were performed using IBM SPSS Statistics for Macintosh, V.25.0. A two-tailed p value of <0.05 was considered statistically significant.Over a 7-year period, 2253 patients underwent cardiac CT imaging at our institution. In all, 500 patients were excluded from the study cohort as they underwent imaging for indications other than investigation of chest pain, or because of a non-diagnostic calcium scan. Therefore, 1753 patients with symptomatic chest pain (mean age 56.8±12.0 years) were included in the study , and 46.A total of 289 patients did not undergo CTCA after CAC scoring (159 patients with a zero CAC and 130 with non-zero CAC). The decision to perform a CTCA after CAC scoring was in line with contemporaneous clinical guidelines and hospital protocol. The major reasons were first, a low pretest probability for CAD plus a zero CAC score (n=112), and second, extensive calcification on CAC scan thought likely to preclude CTCA analysis (n=88). CTCA was not performed in a further 89 patients because of difficult venous access, contrast allergy or patient preference. In addition, subjects with non-interpretable CTCA scans (n=7) were excluded from the CAD extent analysis, but their calcium scores and clinical progress contributed to MACE and mortality estimates.Of the 751 patients with a zero CAC in whom CTCA was performed, 674 (89.7%) had normal coronary arteries, 63 (8.4%) had <50% stenosis and 14 (1.9%) had ≥50% stenosis . In the In those with a zero CAC, there was no significant difference in the baseline risk factors in those with or without obstructive CAD (≥50% stenosis) with the exception of obesity and smoking history .The negative predictive value of a zero CAC for excluding obstructive CAD (≥50% stenosis) was 98.1%. The sensitivity of this was 93.7% and the specificity was 59.7%.Patients were followed up for a median of 2.2 years (IQR 1.8–3.7 years). The longest follow-up period was 7 years. Kaplan-Meier curves for MACE-free survival are presented in MACE in those with zero CAC were due to non-fatal myocardial infarction and/or non-elective revascularisation. None died of a coronary event. MACE occurred at a time interval of 13 days to 1.8 years after cardiac CT. Of the patients who had a zero CAC, three patients had normal coronary arteries and one patient had ≥50% stenosis on CTCA. Of note, 1 patient who had a zero CAC score did not have CTCA and subsequently developed a non-ST elevation myocardial infarction and received non-elective revascularisation at 116 days post-scan.In the unadjusted univariable analysis, there was moderate evidence that the survival distributions for the zero CAC versus the non-zero CAC cohort differed . After adjusting for the confounding factors age and gender, this difference was no longer statistically significant . After adjusting for age, gender plus cardiovascular risk factors, the HR for MACE among the zero CAC cohort vers non-zero CAC was 2.1 .During follow-up, the incidence of all-cause mortality was three patients (0.17%) in the zero CAC cohort versus 20 patients (1.1%) in the non-zero CAC cohort. Annualised rates of all-cause mortality for the zero CAC cohort were 1.2 per 1000 person-years vers 8.7 per 1000 person-years in the non-zero CAC cohort.In the unadjusted univariable analysis of all-cause mortality, there was evidence that the survival distributions for the zero CAC versus the non-zero CAC cohort differed . After adjusting for age and gender, this was no longer statistically significant . After adjusting for age, gender plus cardiovascular risk factors, the HR for all-cause mortality among the zero CAC cohort versus non-zero CAC was 2.1 .A composite endpoint comprising non-fatal myocardial infarction, non-elective revascularisation and/or all-cause mortality resulted in seven patients (0.40%) in the zero CAC cohort versus 36 patients (2.05%) in the non-zero CAC cohort with events. Following univariable analysis, there was evidence that the survival distributions for the zero CAC versus the non-zero CAC cohort differed . This difference remained statistically significant after adjusting for age and gender . After adjusting for age, gender plus cardiovascular risk factors, this was no longer statistically significant .Studies in asymptomatic individuals have shown that the absence of CAC is a reliable screening tool in the evaluation for suspected CAD.10In this prospective cohort study of patients with stable chest pain, a zero calcium score was associated with a low rate of CAD, with 8.4% subjects having non-obstructive disease and only 1.9% found to have obstructive disease. Furthermore, a zero score had a high negative predictive value of 98.1% for excluding obstructive CAD.Our results agree with similar studies in symptomatic patient populations. Among patients with zero CAC in the COroNary CT Angiography Evaluation For Clinical Outcomes: An InteRnational Multicenter Registry (CONFIRM) registry, 13% had at least one <50% stenosis and 3.5% had at least one ≥50% stenosis.14Over a medium-term follow-up, the incidence of MACE and all-cause mortality in our patients was lower in the zero CAC cohort than the non-zero CAC cohort. In multivariable survival analysis after adjusting for age, gender and cardiovascular risk factors, there was no difference in the prognosis between the two cohorts for both MACE and all-cause mortality end points . However, the overall prognosis of stable angina in patients with zero CAC was good with an annualised rate of MACE in the zero CAC cohort of 1.9 per 1000 person-years.We compared both MACE and all-cause mortality in patients with zero CAC versus non-zero CAC, whereas previous studies have focused only on all-cause mortality, and showed this to be 0.05% to 3.8% over a mean follow-up period of 5.6–15 years.12Although subjects with zero CAC had a good prognosis over during medium-term follow-up, 10.3% were found to have CAD on subsequent CTCA. Once identified, this group can benefit prognostically from medical therapy as demonstrated in the Scottish COmputed Tomography of the HEART Trial (SCOTHEART).As an observational study, clinical data were obtained from the electronic records system and there may have been incomplete patient follow-up. We assumed that loss to follow-up would occur equally across the study cohort, thereby not unduly influencing results. As a single-centre study, our findings may not be applicable in other geographic regions. Finally, while we analysed the degree of luminal stenosis on CTCA, we did not take into account the impact of high-risk plaque features that are also known to have prognostic significance.17A zero CAC score in patients with stable chest pain reliably excludes obstructive CAD and is associated with an overall very good prognosis. However, CTCA provides further diagnostic and prognostic information, revealing underlying CAD in over 10% of patients. We now know that such patients should be treated with preventive medical therapy to improve their prognosis.


Information of residue-residue contacts is essential for understanding the mechanism of protein folding, and has been successfully applied as special topological restraints to simplify the conformational sampling in de novo protein structure prediction. Prediction of protein residue contacts has experienced amazingly rapid progresses recently, with prediction accuracy approaching impressively high levels in the past two years. In this work, we introduce a second version of our residue contact predictor, DeepConPred2, which exhibits substantially improved performance and sufficiently reduced running time after model re-optimization and feature updates. When testing on the CASP12 free modeling targets, our program reaches at least the same level of prediction accuracy as the best contact predictors so far and provides information complementary to other state-of-the-art methods in contact-assisted folding. Indeed,Prevalent sequence-based algorithms that predict residue contacts from amino acid sequences can be roughly classified into two categories: methods based on supervised machine learning  and methL/5 predicted contacts rises from 44.0% to 69.6% for the CASP12 targets), and reaches a level comparable to the other state-of-the-art methods including RaptorX-Contact, DNCON2 and SPOT-Contact. On the other hand, the new version was implemented with GPU acceleration, and thus the running time has been sufficiently reduced, especially for large protein targets. Finally, we developed a user friendly online server of DeepConPred2 to provide service for the prediction of residue contact maps.Our previous contact predictor DeepConPred  could re2The flow chart of DeepConPred2 is shown in 2.1We merged the training set and test set used in the study by Xiong et al.  as our nWe prepared an independent test set of 77 protein domains that belonged to novel superfamilies. Specifically, we extracted protein domains newly released in the SCOPe 2.07 version   targets, the hardest targets in de novo protein structure prediction, and then extended the evaluation to all CASP12 targets, including additional 31 template-based modeling (TBM) ones.2.2We updated some input features in the first module. Specifically, we adopted Spider3  secondar2.3Our previous version only processes long-range residue pairs. In the new version, we developed two additional DBN models to predict the contacts between short- and medium-range residue pairs. The short-, medium- and long-range models read in very similar features. However, we updated some features such as coevolutionary information (from CCMpred instead of plmDCA), predicted secondary structure and solvent accessibility (both from Spider3 instead of the previous SSpro and ACCpro ), and so2.4L × L × 9, where L is the length of the protein and 9 can be interpreted as the number of features for one pixel on the contact map (i.e. the target residue pair). We adopted 64 3 × 3 2D convolution filters for each convolutional layer. The convolution stride was set as 1 and the zero-padding pattern was set as ‘same’ to make feature maps of all layers have the same shape as the original input (i.e. L × L). Leaky rectified linear unit (leaky-ReLU) activation function was used for non-linear transformation. The depth of our ResNet models varied from 50 to 80. By stacking many residual blocks, our networks could capture interdependency of residue pairs of very long range even though we used a small filter size.In the third module, we optimized five ResNet models with slight variation of architecture and finally took the ensemble average of these models as the final prediction . The inpDue to the limitation of memory usage when training the ResNet models, length of training input proteins was limited to no >400. For each of the 128 protein domains that exceed this limit, we randomly chose 4 overlapping 400 × 400 subplots as input.2.5All parameters were optimized using 5-fold cross validation on the training set. In this process, we adopted the F1-score for all available residue pairs to evaluate individual models. The F1-score is defined as the harmonic mean of Precision and Recall:β atoms falling within 8.0 Å were considered to be in contact. Following the CASP routine, we chose the precisions , RaptorX-Contact (http://raptorx.uchicago.edu/ContactMap/) and SPOT-Contact (http://sparks-lab.org/jack/server/SPOT-Contact/) were obtained from their web servers in July 2018, respectively. Then, we extended the comparison to the overall 53 CASP12 targets that have available structure information, including additional 31 TBM ones. Results of additional targets were received on October 5th, 2018 from the online servers of RaptorX-Contact and SPOT-Contact. The server of DNCON2 was unavailable for new predictions at that time for some reasons. Thus, we had to use the latest downloadable results of the CASP12 test posted on the website, which only contained 35 targets.In this work, we compared our method with three state-of-the-art algorithms, DNCON2, RaptorX-Contact and SPOT-Contact. The comparison was first performed on 22 CASP12 FM targets that have available structure information. Prediction results of DNCON2  to the native structure) among the top 5 ones as reported by CONFOLD was taken as the representative structural model of each target for various contact predictors.To evaluate the contribution of contact predictors in protein structure prediction, we modeled the structures of the 22 CASP12 FM protein targets  using the top 2 program  to proce33.1In the first and second modules, we only retained the best DBN models based on F1-scores in the 5-fold cross validation. In the third module, however, we constructed ResNet models with various levels of depths. In the final prediction, ensemble average of these models was adopted to further improve the performance .t, G, f and Err represent expectation, the t-th model in the ensemble, ensemble average, the truth ground (label) and model error, respectively. In this work, F1-score of the ensemble (0.5711) surpasses all individual models.Average ensemble is also used in RaptorX-Contact and SPOT-Contact ,18, whic3.2L/5 predicted long-range residue pairs reaches 40.61%, 43.35% and 44.00% on the independent test set, CASP11 set and CASP12 set respectively for the old version, but rises to 70.67%, 71.26% and 69.60% respectively in the new version. Balanced performance of our new program on the three testing sets also supports robustness of this method for different kinds of targets.We evaluated the performance of DeepConPred2 over the previous version on three testing sets. The previous version can only predict contacts of long-range residue pairs while the new version has no such limits. As shown in Generally, in comparison to the old version, the new version mainly includes modifications on three facets: A) updates of input features and architecture fine-tuning of the DBN models in the first and second module; B) consideration of short- and medium-range predictions in the second module; and C) adoption of ResNet in the third module. We roughly estimated their contributions to the performance improvement for long-range residue contacts on the independent test set. As shown in Table S1, modification A brings the largest improvement in prediction accuracy (by ~16 percentage points), followed by modification C, which contributes by ~10 percentage points, while modification B only makes a minor contribution.Moreover, the new version greatly reduces the noise levels in the predicted residue contact maps. For an example target, the previous version generates a contact map with serious palisade noises that cover useful contact information a. In ourIn addition to the substantial improvement in prediction accuracy and map quality, the new version was implemented using the TensorFlow framework  with GPUTo provide convenient service for residue contact prediction, we also constructed a web server of DeepConPred2. Users could submit the protein sequence on the website and approach the prediction results later by email notification. Meanwhile, program for local execution is also provided for free download.3.3L/5 precision: 57.56% vs. 53.49%). When compared with RaptorX-Contact, DeepConPred2 shows similar performance for long-range contacts. For example, the long-range top L, L/2 and L/5 precisions of DeepConPred2 vs. RaptorX-Contact are 41.27% vs. 39.50%, 49.16% vs. 51.15% and 57.56% vs. 58.55%, respectively. As for short- and medium-range contacts, DeepConPred2 exhibits an advantage to RaptorX-Contact. For medium-range contacts, the top L, L/2 and L/5 precisions of DeepConPred2 vs. RaptorX-Contact are 30.78% vs. 23.64%, 42.63% vs. 36.08% and 56.87% vs. 53.41%, respectively, while for short-range contacts, they are 31.90% vs. 22.57%, 44.34% vs. 35.83% and 58.49% vs. 57.20%, respectively. DeepConPred2 is slightly prevailed by SPOT-Contact .We first compare our method with three state-of-the-art algorithms, DNCON2, RaptorX-Contact and SPOT-Contact, on 22 CASP12 FM targets. As shown in We then extended the comparison to all 53 CASP12 targets that have available structure information (Tables S2–S3). Because DNCON2 server was temporarily unavailable for prediction, comparison with DNCON2 was only conducted on 35 targets that have downloadable prediction results posted on the DNCON2 website. Similar to the previous comparison, DeepConPred2 significantly outperforms DNCON2, but is slightly prevailed by SPOT-Contact. Possibly due to the strong prediction power on TBM targets, the relatively easy targets for de novo protein structure prediction, performance of RaptorX-Contact slightly surpasses DeepConPred2 and becomes close to SPOT-Contact. Nevertheless, DeepConPred2 reaches a high level of prediction accuracy  and thus can be ranked as one of the best residue contact predictors. It should be noticed that updates of sequence databases, especially the introduction of metagenome sequence data, would have big impact on performance of predictors . It is p3.4L predicted contacts as the only constraints. For each protein, we chose the one with minimum RMSD to the native structure from the top 5 models provided by CONFOLD as its representative model for the corresponding algorithm. We performed Levene test to make sure the equal variance between RMSD groups of different methods and then conducted 2-sided paired t-test to check whether there are any significant differences. At the confidence threshold of 0.1, CONFOLD models using contact prediction of our method surpass those using DNCON2 significantly but have no significant differences from those of RaptorX-Contact or SPOT-Contact . Deming regression and Passing-Bablock regression analyses (Tables S7–S9) also indicate that the four methods have the same level of powers on contact-assisted folding.Despite the CASP12 report that usage of coevolutionary information and ultra-deep learning framework has raised accuracy performance of contact prediction into a new level , our ana4In this work, we present a second version of our residue contact predictor, DeepConPred2, which can predict all contacts of a protein at once from a protein sequence. The new version exhibits remarkably improved performance in comparison to the previous one, and could reach a prediction power comparable to the other state-of-the-art methods. In addition, implementation with GPU acceleration further reduces the running time, especially for large protein targets. A user friendly web server is available for reliable prediction of residue contact maps.


Bone marrow mesenchymal stem cells (BM-MSCs) elicit neuroprotective effects, and their repair ability has been investigated in different experimental models. We aimed to investigate the effect of multiple i.p. BM-MSCs injections in the cuprizone model of multiple sclerosis in mice.6 in 1 milliliter medium) were administered intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. Animals (n = 12) were perfused with 10% paraformaldehyde at the end of sixth week. The brains were sectioned coronally in 6-8-μm thickness (-2.3 to 1.8 mm from bregma). The sections were stained by luxol fast blue-cresyl violet, and images were captured via a microscope. Demyelination ratio was estimated in corpus callosum in a blind manner. A quantitative real-time PCR was used to measure the myelin basic protein gene expression at sixth week.Adult male C57BL/6 mice (n = 40) were fed a regular diet or a diet containing cuprizone (0.2% w/w) for six weeks. Bone marrow samples were taken from patients with spinal cord injury. BM-MSCs (2 × 10Histologically, cuprizone induced demyelination in the corpus callosum. Demyelinated area was diminished in the corpus callosum of cell-administered group. Cuprizone could decrease myelin-binding protein mRNAs expression in corpus callosum, which was significantly recovered after BM-MSCs injections.Our data indicated a remyelination potency of multiple i.p. BM-MSCs in the cuprizone model of multiple sclerosis in mice. Multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS) with demyelination and gliosis, can affect young individuals. RemyeliEvidence has indicated the repairing effect of stem cell transplantation in demyelination. It has BM-MSCs have been suggested to enhance the restoration of oligodendrocytes and therefore remyelination. These cConsidering the aforementioned aspects, the i.v. transplantation of BM-MSCs can reduce their efficacy and their migration potency, decline the general cell survival and enhance danger of undesired influence. The i.p. transplantation is effectively a safe and a further efficacious way of BM-MSCs administration for disorders within the abdominal cavity. The i.p. injection of stem cell transplantation is easy to carry out and has the outlook of extensive choice in clinical cell therapy and has a high impact on patient management. The i.p. injection of BM-MSCs has been shown to be significantly more effective than i.v. administration for ameliorating clinical signs in a rodent model of inflammatory bowel disease. The i.pMale C57Bl/6 mice (n = 40), 7-8 weeks old, were purchased from Pasteur Institute of Iran (Tehran). The animals were subjected to a 12:12 h daylight/darkness with free access to food and water. The procedures were performed in accordance with institutional guidelines for animal care and use. The Research and Ethics Committee of the School of Advanced Technologies in Medicine, Tehran University of Medical Sciences approved the experimental protocol (project number 93-03-87-25963). The mice were fed with cuprizone  in powdered rodent lab chow for a period of six consecutive weeks, as described previously. An addi2, 85% humidity, at 37 °C. After 2 days of initial culture, non-adherent cells were removed, and adherent cells (80-90% confluence) were passaged. Medium replacement was done every 3-4 days. Grown colonies of adherent cells were separated with 0.025% Trypsin-EDTA and subcultured at a density of 2,000 to 5,000 cells/cm[Human MSCs were generated by conventional lab procedures: after obtaining an informed consent from the patients, the cells were harvested from the iliac crest during stem cell therapy for spinal cord injury patients. Bone marrow aspirated tissue was diluted with 4 volumes of PBS, filtered and centrifuged to density gradient with ficoll. The mononuclear cells were disported from the interface and washed in PBS, then suspended in a medium and put into 25-cm cell culture flasks. The medium contained alpha-minimum essential medium (α-MEM) supplemented with 10% (vol/vol) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were cultured in 5% CO cells/cm. Cells w6, the cells were incubated with 20 μl FITC-conjugated monoclonal antibody for CD73 or with phycoerythrin (PE)-conjugated monoclonal antibodies for CD34, CD45, CD90, CD105, CD13, and CD49e (1 h at room temperature). Isotype control antibodies were utilized for deletion of any non-specific reaction. FACS analysis was performed with a FACScalibur flow cytometer [Cells were analyzed for the expression of BM-MSCs markers after forth passage using flow cytometry The Cultivated cells were trypsinized and washed with PBS. At a density of 5 × 10Cells were incubated in α-MEM containing 20% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 12 mM L-glutamine, 5 mg/ml insulin (Sigma-Aldrich), 50 mM indomethacin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), and 0.5 mM 3-isobutyl-1-methylxanthine for 2 weeks. Cells were then fixed with 10% formalin at room temperature for 20 min and stained with 0.5% Oil Red O (Sigma-Aldrich) in methanol (Sigma-Aldrich) at room temperature for 20 min .4 cells per well in BM-MSCs medium. After 24 h, the medium was changed with osteogenic medium . After ten days, samples were stained with Alizarin Red S[BM-MSCs were cultured in multi-well plates at 3 × 10rin Red S.th passage of BM-MSCs, slides of prometaphase chromosomes were prepared with a hypotonic solution  at 37.5 °C for 15 min and fixed with methanol-acetic acid solution for 1 h. The cell slides were incubated in quinacrine mustard solution at room temperature for 10 minutes, rinsed with Sorensen’s buffer (pH 6.8) and mounted in the same buffer. The slides were studied via a fluorescence microscope[In the 6icroscope.6 BM-MSCs in 500 µl of PBS intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. All experimental groups were kept on the cuprizone diet for an additional two weeks, following the i.p. administration of either PBS (sham) or BM-MSCs.Animals were administered 2 × 10At the end of sixth week of cuprizone diet, animals (n = 12) were perfused with 10% paraformaldehyde for immunohistochemistry and histology assays. Coronal brain slices (8 µm) between -2.3 to 1.8 mm from bregma were prepared according to Paxinos and Franklin’s stereotaxic atlas. SectionSYBR Green . The double delta Ct analysis procedure was utilized to determine differences in the expression of MBP gene between animal groups. Survey in the mRNA expression quantity was computed after normalization to β-actin[Quantitative real-time PCR was performed to quantify the level of MBP gene expression in corpus callosum. RNA from tissues was prepared following the manufacturer’s specifications . cDNA was synthesized using the High-Capacity cDNA Reverse Transcription . Real-time quantitative PCR analysis was performed using StepOne™ Real-Time PCR System equipped with the o β-actin. The prip < 0.01 was considered as statistically significant.All data were expressed as the mean ± SD. Statistical analysis included one-way ANOVA and Duncan post-hoc test for post hoc comparison if appropriate. Fibroblastic cells began to appear in the culture flasks five to seven days after plating bone marrow nucleated cells. The non-adherent hematopoietic cells in the culture were removed during the changes of medium. Initially, fibroblastic cells in a single colony were often separated from each other ; howeverAfter adipogenic induction, the cell morphology was changed from the elongated confluent fibroblastic cells to more oval shaped cells, which showed a distinct ring of red coarse vacuoles around the cell periphery after Oil Red O staining. These vacuoles appeared to be developed by day two and became more numerous and larger with time .While growing in the osteogenic medium, BM-MSCs tend to aggregate and make “knotted formation”, which is visible by microscope. Mineralization of the aggregate is usually reported by compacted, refrangible sediment, which was assayed by measuring calcium deposition via Alizarin Red S staining .The cells from passages four were tested by FACS analysis for the expression of the mesenchymal cells markers . More than 80% of the BM-MSCs derived from the bone marrow stem cell populations expressed the typical BM-MSCs marker proteins CD90, CD73, CD13, CD49e, and CD105. Also, more than 90% of the cells were negative for CD34 and CD45 .6 cells/500 µl of PBS, i.p.) or an equivalent volume of PBS (sham) for two consecutive weeks, which was started at the end of the forth weeks of cuprizone administration. Staining of myelin with luxol fast blue displayed a steady and a profound loss of myelin within the corpus callosum of cuprizone exposed mice in the model and in the sham treatment groups, as compared to the stem cell-treated mice (p < 0.01). BM-MSCs treatment provided a significant reduction in the demyelinating effects of cuprizone (p < 0.01) although demyelination was not completely remyelinated (p < 0.01). To investigate the probable mechanisms by which BM-MSCs restorate cuprizone-induced demyelination, we studied whether the i.p. administration of BM-MSCs migrates from the i.p. cavity to the brains of the injected animals. The DiI-labelled transplanted BM-MSCs were not observed in corpus callosum of the injected group.Figures ted mice . This anp < 0.05) in comparison with the model and the sham groups. We did not observe any difference in the expression of house-keeping gene, β-actin, among the experimental groups. Three mice were used for each group. The qRT-PCR data were calculated as normalized relative quantity (RQ) values, with all RQ values for a gene being normalized to the mean RQ value of the respective model mice.To explore the hypothesis that an alteration in MBP function is present in cuprizone-induced demyelination, we investigated the mRNA expression level of MBP using real-time PCR in the corpus callosum. The data analyses for the normalized expression of MBP gene are shown in Previous experiments using BM-MSCs in several models of multiple sclerosis led us to use the cuprizone model of multiple sclerosis. Since the cuprizone model is in drastic conflict to human multiple sclerosis, it does not mirror the complicated pathophysiology of the human multiple sclerosis where the peripheral immune system acts as a major player in injury propagation. Neverthet al.[In the present study, we showed that the i.p.- administered BM-MSCs did not migrate to corpus callosum but improved myelination in this part of brain. However, another study showed that i.v. or i.p. injections result in rapid reduction in cell numbers. Few studies have also indicated the effect of mesenchymal stem cells on multiple sclerosis animal models. Thus, aet al. have demOur present results indicated that the i.p. administration of BM-MSCs improves myelination as it is shown in luxul fast blue staining and also increases MBP gene expression. In this study, we found that BM-MSCs did not migrate to injured site by DiI staining. However, these cells could significantly affect remyelination. Similarly, a previous study proved the potency of intracranially transplanted mesenchymal stromal cell to strengthen endogenous repair in chronic cuprizone-treated mice. OligodeWe did not detect any BM-MSCs in rostral corpus callosum with i.p. administration. However, this route of administration elicited remyelination in the evaluated regions. Similarly, in EAE model, in which the peripheral immune system has a great role, stem cell administration suppressed the disease. TherefoMBP is an abundant component of the oligodendrocyte myelin membrane, alternatively spliced from the Golli-MBP gene. MBP is critical to myelin membrane biogenesis and to regulation of the entry of other proteins into the membrane sheets. Our resWe utilized multiple i.p. injections of BM-MSCs in the cuprizone model of multiple sclerosis and showed that this method can induce remyelination within two weeks. A reasonable therapeutic method of multiple sclerosis must consider all the pathophysiological processes of the disease and must be multipurpose. In establishing EAE model, BM-MSCs have demonstrated their potency in supplying an immunomodulatory action. Nevertheless, in other models of demyelination, which are not related to immune system, these cells can have beneficial ability to repair demyelination through immune-independent process. To the best of our knowledge, this is the first experiment demonstrating that multiple i.p. injection of BM-MSCs can ameliorate demyelination in the cuprizone model of multiple sclerosis through stimulating remyelination process.


Adverse early life stress is a major cause of vulnerability to various mental disorders in adulthood, including post-traumatic stress disorder (PTSD). Recent studies have suggested that early life stress can help the body adapt optimally when faced with stressful trauma in adult life. An interaction may exist between early life stress  and vulnerability to PTSD. This study aimed to evaluate emotion-related behaviors and verify the long-lasting effects of cognitive aspects of PTSD after exposure to severe adverse early life stress, such as long-term separation. Adverse early life stress was simulated by subjecting rats to 3 or 6 consecutive hours of maternal separation (MS) daily, from postnatal day (PND) 2 to PND 14. Single-prolonged stress (SPS) was simulated on PND 80 to imitate other adulthood stresses of PTSD with gender divisions . After the MS and PTSD sessions, behavioral tests were conducted to assess the effectiveness of these treatments, which included an open field test (OFT), elevated plus maze test (EPMT), water maze test (WMT), and forced swimming test (FST) to detect anxiety-like behavior (OFT and EPMT), memory behavior (WMT), and depressive behavior (FST). The M-MS3h-PTSD group had fewer time entries into the open arms of EPMT than the F-MS3h-PTSD group, and the M-MS6h-PTSD group demonstrated fewer up-right postures in the OFT than the F-MS6h-PTSD group. The M-MS3h-PTSD group exhibited more exploratory behavior than the M-MS6h-PTSD and M-PTSD groups in the OFT. Less exploratory behavior was observed in the F-MS3h-PTSD group than in the F-MS6h-PTSD group, which demonstrated significantly increased freezing times in the FST compared to the F-PTSD group. The WMT revealed significant differences in learning and memory performance between the M-MS3h-PTSD group and other treatment groups, which were not found in the female rats. These findings demonstrate that an early stressful experience, such as MS, may be involved in helping the body adapt optimally when faced with additional trauma in adulthood, although mild early life stress might benefit learning and memory among males. Studies have found that regions of the brain that are especially susceptible to stress during the first 7 years of life –3 are inEarly postnatal stress animal models have been developed due to the high prevalence and serious consequences of early stress in humans. Rats exposed to early-life stresses, such as maternal separation (MS) during lactation, serve as good models for studying the effects that neglect has in early life . The usePost-traumatic stress disorder (PTSD) is a highly disabling condition observed in individuals following exposure to severe emotional or physically life-threatening traumatic events , compounRepeated MS is thought to increase animals’ sensitivity to stress . Some stThe aim of the present study was to compare the functional consequences of the PTSD model after MS at either 3h or 6h every day over a wide range of behaviors, which were either emotion-related (anxiety and depression) or learning-related  in adulthood. The PTSD model after MS in rats imitates orphans or left-behind children exposed to a traumatic event (or events), as it alters vulnerability to psychiatric disorders and cognitive deficits in adult life. The sex of rats was considered because sexual differences in enduring early-life stress could induce rodents’ alterations in neurogenesis .Pregnant Sprague–Dawley (SD) rats were bred at our animal facility and randomly selected from the Animal Center of Weifang Medical University. The day of birth was recorded as postnatal day (PND) 0. Pups were subjected to either MS or animal-facility-reared treatment . Rats wen = 7), male rats with PTSD , male rats with 3h MS and PTSD , male rats with 6h MS and PTSD , female control group , female rats with PTSD , female rats with 3h MS and PTSD , and female rats with 6h MS and PTSD . In the F/M-MS3h-PTSD groups, litters were separated from their dams for 3h (9:00–12:00) each day  procedures were similar to previous experiments , conductAll animals underwent all behavioral experiments, which were conducted sequentially with the same sequences: 1) open field test (OFT), 2) elevated plus maze test (EPMT), 3) water maze test (WMT), and 4) forced swimming test (FST) Figure 1Open field test. The chamber was a box without a lid (100 × 100 × 50 cm) consisting of a black floor and walls. Incandescent bulbs installed on the ceiling were used for illuminating the box. Each rat was transported from the colony room to the OFT room on the day of OFT. At the beginning of the experiment, the animals were placed gently in the corner and allowed to explore the chamber freely for 5 min, and upright numbers were recorded at the same time. Automated image analysis software  was used for recording time spent in central areas, which evaluated the anxiety-like behavior. The open field was cleaned with ethanol after each test.Elevated plus maze test. The EPMT is commonly used to score PTSD-like anxiogenic behavior in rats by exploiting the conflict between rats’ innate fear of open spaces versus their desire to explore novel environments  with Tukey. Independent-samples t-test was used for the comparison of gender difference. A repeated-measures multivariate analysis of variance (MANOVA) was conducted to analyze of WMT escape latencies using SPSS statistical software  at a significance level of F = 3.88, p = .025; Post hoc test: p = .017). The M-MS3h-PTSD and F-MS3h-PTSD rats showed no obvious gender differences between upright times  = 3.97, p = .023, Post hoc test: p = .026; F = 3.59, p = .032, Post hoc test: p = .028),  and number of entries into open arms of EPMT ,  = 10.92, p < .001). The results showed that the separate effect that the treatment had was significant from day 1 to day 3 . On day 2, the PTSD and MS6h-PTSD groups demonstrated more time compared to the control  and MS3h-PTSD  groups. On day 3, the MS3h-PTSD group used less time compared to the control (p = .003) and PTSD (p = .003) groups. However, there were no striking differences on day 4 between any of the treatment groups. In female rats the interaction between treatment and time was statistically significant  = 12.73, p < .001). Further analysis of the female groups showed that the separate effects of treatment was significant on day 1, day 3 and day 4  apart from that MS6h-PTSD group had significantly less time compared to the PTSD (p = .004) and MS3h-PTSD group (p = .001). No striking difference was found on day 2 between any of the groups. On day 3, the PTSD group spent less time compared to other groups , and the MS3h-PTSD group used less time compared to the MS6h-PTSD group (p = .006). On day 4, the MS3h-PTSD group used less time compared to the control group (p = .001) as well as the MS6h-PTSD group (p = .010), and the MS6h-PTSD group used significantly more time compared to the PTSD group (p < .001). When we explored rats controlling for gender difference, the interaction was statistically significant between treatment and time  = 16.16, p < .001). The separate effects of treatment were significant from day 1 to day 4 . On day 2, the PTSD group used more time compared to the control group (p = .005) and the MS3h-PTSD group (p < .001). On day 3, the control group required more time compared to the PTSD group (p = .017) and the MS3h-PTSD group (p = .003), and the MS6h-PTSD group used more time compared to the PTSD group (p = .027) and the MS3h-PTSD group (p = .005). On day 4, the control and MS6h-PTSD groups used more time respectively compared to the PTSD group  and the MS3h-PTSD group .A repeated MANOVA was used to analyze the mean latency of each group in finding the platform during initial spatial learning with treatment and test days across different genders and separation time of MS. In male rats, the interaction was statistically significant between treatment and time , female  or from the group without gender difference , suggesting that the groups’ learning ability improved after the treatment test.The separate effect that the test day had from day 1 to day 4 was significant in all treatments no matter if the rats were male  = 2.72, p = .034). No significant differences were found between treatments  = 8.49, p = .001; Post hoc test: p = .001) and no immobility difference in male rats  = 8.49, p = .002). No significant differences emerged in depressive-like behavior between the MS3h-PTSD, MS6h-PTSD, and PTSD groups when there was no gender distinction  , 39. CarSeveral animal models of PTSD, such as those involving social stress and predator stress, have been proposed. These animal models fail to demonstrate the most consistent neuroendocrinologic characteristic observed in PTSD patients, namely enhanced inhibition of the HPA axis, although they present behavioral alterations like PTSD –44. The via EPMT , the science and technology foundation program of the colleges and universities of Shandong province (J17KB096), and the research award foundation program for outstanding young scientists of Shandong province (BS2014YY043), and the Students’ Research Fund of Weifang Medical University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.